A Synthetic Biology Approach for the Characterization of the Inositide Signaling Pathway, and Characterization of an IP6 binding Protein
Clarke, Bradley Palmer
Myo-Inositol is the precursor to a family of molecules called inositides. The two major classes of inositides, the lipid phosphatidylinositol phosphates (PIPs) and the water-soluble inositol phosphates (IPs) are conserved throughout eukaryotes and are required for life. This dissertation describes both a synthetic biology-based approach to studying the inositide pathway as a whole and the biochemical and structural characterization of a protein, Clu1, that binds to inositol hexakisphosphate (IP6). Deconvoluting the metabolic and functional aspects of inositide biology can be confounded by the promiscuity and multiplicity of the PIP and IP kinases and phosphatases. Here we describe a synthetic biology-based system that reconstitutes the eukaryotic PIP and IP pathways in Escherichia coli, which inherently lack inositide kinases and phosphatases. By expressing cassettes of eukaryotic inositide modifying enzymes in bacteria, we show that the key components of inositide synthesis can be reconstituted. By studying inositide biology in a synthetic system, heretofore unappreciated activities can be observed, including an alternative pathway to produce PIP3. Additionally, this bacterial system can be utilized for the reconstitution of lipid dependent synthesis of IPs, including IP6. While many inositides serve as signaling molecules, there is a growing class of proteins that bind IP6 as a structural cofactor. This work describes the biochemical and structural characterization of one IP6 binding protein, Clu1. Clu1 is a protein that is required for the correct distribution of mitochondria, plays a role in responding to mitochondrial damage, and that binds to mitochondrially targeted mRNAs. We show that the RNA binding of Clu1 is mediated by its C-terminal 50 amino acids and that RNA binding is separate from IP6 binding. Using a variety of techniques, we demonstrate that Clu1 is a dimer with an elongated shape, and that there are two conformations of the Clu1 homodimer. We also report the characterization of Clu1 using X-Ray crystallography and cryo electron microscopy. This work describes two different approaches for the study of inositides, and the function of IP6 though its mediation of the protein Clu1.